【 For USA 】

Cat. # 6230, 6652, 6655

Product Manual

AAVpro® Helper Free System

For Research Use

v201611Da

Table of Contents

I. Description........................................................................................................ 4

II. Components.................................................................................................... 6

III. Storage............................................................................................................... 9

IV. MaterialsRequiredbutnotProvided..................................................... 9

V. OverviewofAAVParticlePreparation..................................................10

VI. Protocol............................................................................................................10

VII. MeasurementofVirusTiter......................................................................12

VIII. ReferenceData..............................................................................................13

IX. References.......................................................................................................17

X. RelatedProducts..........................................................................................17

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Safety & Handling of Adeno-Associated Virus VectorsTheprotocolsinthisusermanualrequirethehandlingofadeno-associatedvirusvectors.Itisimperativetofullyunderstandthepotentialhazardsofandnecessaryprecautionsforlaboratoryuseofthesevectors.VirusesproducedwithAAV-basedvectorscould,dependingonyourgeneinsert,bepotentiallyhazardous.Similarvectorshavebeenapprovedforhumangenetherapytrials,attestingtotheirpotentialabilitytoexpressgenesinvivo.Forthesereasons,duecautionmustbeexercisedintheproductionandhandlingofanyrecombinantviruses.FollowallapplicableguidelinesforresearchinvolvingrecombinantDNA.Takeappropriatesafetymeasureswhenproducingorhandlingrecombinantadeno-associatedviruses,includingworkinginabiologicalsafetycabinetandwearingprotectivelaboratorycoats,faceprotection,andgloves.

Available AAVpro ProductsAAVpro®PurificationKit(AllSerotypes) Cat.#6666AAVpro®PurificationKit(AAV2) Cat.#6232AAVpro®TitrationKit(forRealTimePCR)Ver.2 Cat.#6233AAVpro®PackagingPlasmid(AAV2) Cat.#6234AAVpro®ExtractionSolution Cat.#6235pAAV-ZsGreen1Vector Cat.#6231AAVpro®293TCellLine Cat.#632273

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I. DescriptionI-1. Adeno-Associated VirusAdeno-AssociatedVirus(AAV)isanon-envelopedvirusthatbelongstotheParvovirusfamilyoftheDependovirusgenus.AAVisnotthoughttobepathogenictohumansandonlyreplicatesinthepresenceofahelpervirus,suchasadenovirusorherpesvirus.TheAAVgenomeisalinear,single-strandDNAmoleculeofapproximately4.7kbthathasterminalhairpinstructurescalledinvertedterminalrepeats(ITRs)atbothends.ITRsfunctionasoriginsofgenomicreplicationandcontributetopackagingofviralparticles.TheAAVgenomeincludesthreeopenreadingframes(Figure1):Rep,whichencodesaproteininvolvedinreplicationandtranscription;Cap,whichencodescapsidproteins;andAAP,whichencodesanon-structuralproteinnecessaryforformationofviralparticles.TheRepregioncodesfor4differentproteins(Rep78,Rep68,Rep52,andRep40),andtheCapregioncodesfor3differentproteins(VP1,VP2,andVP3).Therearemorethan100serotypesofAAV,andthehostspecificityandcharacteristicsofthevirusdifferamongserotypes.Serotype2(AAV2)isthemostcommonlyusedserotypeinAAV-basedresearch,includinggenetherapy,andischaracterizedbyabroadhostrange.Adeno-associatedvirusvectors(AAVvectors)exploitthepropertiesofAAVfortransductionofgenestocellsandorganisms.AAVvectorsareusedasresearchtoolsandalsoasvectorsforgenetherapy.Inaddition,AAVvectorsaregenerallyconsideredsaferthanadenoviralandretroviralvectors.AAVvectorscanbeusedtotransducegenesintobothproliferatingandnon-proliferatingcellsandcanimpartlong-termexpressioninnon-dividingcells.Inaddition,AAVhaslittleimmunogenicityandissuitableforthetransductionofgenesintoanimals(asaninvivotransductiontool).

Figure1. AAVgenomestructureandopenreadingframes.

Rep

ITR

Cap

Rep78

Rep68

Rep52

Rep40

VP1

VP2

VP3

AAP

ITR

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l-2. PrinciplesTheAAVproHelperFreeSystemenablesthepreparationofserotypeAAV2particleswithouttheuseofahelpervirus.TheAAV2particlesproducedcanbeusedtoobtaintransientexpressionofthetargetgeneinmammaliancellsorindividualanimals.Forinvivoapplications,purificationofAAVvectorwiththeAAVproPurificationKit(AAV2)(Cat.#6232)orAAVproPurificationKit(AllSerotypes)(Cat.#6666)isrecommended.

l-3. FeaturesA. Preparation of AAV2 Vector with the AAV Helper-Free SystemTheAAVHelperFreeSystemisauniquesystemforthepreparationofhigh-titerAAVparticleswithouttheuseofahelpervirus(Figure2).EachkitincludesthreeplasmidsencodingthefactorsnecessarytopreparerecombinantAAVparticlesbytransfectionintoHEK293cells.

•pRC2Vector:PlasmidthatexpressestheAAV2RepgeneandtheCapgene

pRC2-mi342alsoexpresseshsa-miR-342,ahumanmicroRNAthatincreasesAAV2titerinvectorpreparationsystems.Itincreasestiterbyapproximately2-foldascomparedtoordinarypRC2vectorsthatexpressonlyRepandCap(VIII.ReferenceData).

•pHelperVector:PlasmidthatexpressesadenovirusesE2A,E4,andVA

•pAAVVector:PlasmidcontainingapromoterforgeneexpressionandtwoITRs

pAAV-CMVVector(Cat.#6230)Plasmidcontainsasiteforcloningageneofinterest(GOI).TheGOIisexpressedfromaCMVpromoter.ThesizeoftheGOIclonedintothepAAV-CMVvectorshouldbe

Figure2. PreparationofAAVparticlesusingtheAAVproHelperFreeSystem.

pAAV Vector pRC2 Vector pHelper Vector

Transcription and TranslationTransfection

Replication

AAV ssDNA

AAV Extrac on Solu on

AAV2 Particles

293 Cell

II. ComponentsEachkitincludesthreeplasmidsencodingthefactorsnecessarytopreparerecombinantAAVparticles,andreagentsforextractingAAVparticlesfromproducercells.AAVproHelperFreeSystem(AAV2)(Cat.#6230)1. pAAV-CMVVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3

AAVproHelperFreeSystem(AAV2-CRERecombinase)(Cat.#6652)1. pAAV-CRERecombinaseVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3

AAVproHelperFreeSystem(AAV2-LacZ)(Cat.#6655)1. pAAV-LacZVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3

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AAVpro® Helper Free SystemCat. #6230, 6652, 6655

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[Plasmids for transfection also sold separately]AAVproPackagingPlasmid(AAV2)(Cat.#6234)1.pRC2-mi342Vector(1μg/μl) 0.5mlx22.pHelperVector(1μg/μl) 0.5mlx2

[Vector maps]

Ampr

pUCori

ITR

ITR

MCS

CMV promoter

β-globinintron

hGHpolyA

pAAV-CMV Vector(5,031 bp)

EcoR VHind III

EcoR VAfl IISfi I

Figure3. pAAV-CMVvectormapandmultiplecloningsite(MCS).

1471 GAATTGGGATTCGCGAGAATTCTCTAGAGTCGACACTAGTGCGGATCCAC 1520 CTTAACCCTAAGCGCTCTTAAGAGATCTCAGCTGTGATCACGCCTAGGTG

EcoR I

Xba INru I Spe I

Sal I Acc IHinc I BamH I

β-globinintron

MCS

Ampr

pUCori

ITR

ITR

CMVpromoter

Humanβ-globinintron

Cre recombinase

Nuclearlocalizationsignal (NLS)

pAAV-CRE RecombinaseVector(6,115 bp)

hGHpolyA

Figure4. pAAV-CRERecombinasevectormap.

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Cat. #6230, 6652, 6655v201611Da

Figure5. pAAV-LacZvectormap.

Figure6. pRC2-mi342vectormap.

Ampr

ColE1ori AAV2

Rep

AAV2Cap

CMVpromoter

hsa-miR-342

HSV-TKpolyA

pRC2-mi342 Vector(8,189 bp)

Ampr

pUCori ITR

ITR

CMVpromoter

LacZ

hGHpolyA

pAAV-LacZ Vector(7,622 bp)

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AAVpro® Helper Free SystemCat. #6230, 6652, 6655

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Figure7. pHelpervectormap.

Ampr

ColE1ori

AdenovirusVA

AdenovirusE4

AdenovirusE2A

pHelper Vector(11,635 bp)

III. Storage-20℃StoreAAVExtractionSolutionAandAAVExtractionSolutionBatroomtemperatureafterthawing.Usewithin2yearsofreceipt.

IV. Materials Required but Not ProvidedIV-1. Equipment

• 100-mmdiametertissueculture-treateddishes• Generalequipmentforcellculture

IV-2. Reagents• TransfectionreagentCalPhos™MammalianTransfectionKit(Cat.#631312)Xfect™TransfectionReagent(Cat.#631317)

• Dulbecco'sModifiedEagle'sMedium(DMEM)4.5g/LGlucosewithL-Glutamine• FetalBovineSerum(FBS)• Trypsin-EDTA• AAVpro293TCellLine(Cat.#632273)*1• 0.5MEDTA(pH8.0)[EDTABufferPowder,pH8.0(Cat.#T9191)]• pAAV-ZsGreen1Vector(Cat.#6231)*2

*1 SeveralHEK293andHEK293Tcelllinesarecommerciallyavailable.Viralproductionishighlydependentonfeaturesofthecellline.AAVpro293TCellLine(Cat.#632273)andHEK293T/17cells(ATCC,CRL-11268)arerecommendedforpreparationofhigh-titerAAV.

*2 pAAV-ZsGreen1VectorisanAAVvectorplasmidthatexpressesthegreenfluorescentproteinZsGreen.UseasapositivecontroltoconfirmtheefficiencyoftransfectionandthetiterofthepreparedAAVparticles.

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V. Overview of AAV Particle PreparationPerformallstepsfromstepVI-1forAAVproHelperFreeSystem(AAV2)(Cat.#6230)tocloneaGOI.Fortheothersystems(Cat.#6652and6655),performstepsVI-3throughVI-7.

1. Cloningageneofinterest(GOI)intopAAV-CMVVector↓

2. Preparetherecombinantplasmid(pAAV-GOIvector)↓

3. CultureAAVpro293Tcells↓

4. Co-transfectAAVpro293TcellswithpAAV-GOI,pRC2-mi342,andpHelpervectors↓

5. Changeculturemedium↓

6. CollectAAV-producingcells(2-3daysaftertransfection)↓

7. ExtractvirusparticlesfromAAV-producingcells

VI. ProtocolVI-1. Cloning a Gene of Interest into pAAV-CMV Vector

Insertageneofinterest(GOI)intothemultiplecloningsite(MCS)ofthepAAV-CMVvectorusingstandardcloningmethods.TheIn-Fusion®HDCloningPluskit(Cat.#638909)canalsobeusedtoeasilyclonePCRproductsderivedfromtheGOIintoanylinearizedvector.Inaddition,theCMVpromotercanbereplacedwithanotherpromoterusingtheEcoRVsiteinthisplasmid(Figure3).

Note 1: TheGOIDNAfragmentshouldcontainanATGstartcodonandastopcodon.

Note 2: ThesizeoftheGOIinsertshouldbe

VI-4. Transfection of AAVpro 293T Cells with pAAV, pRC, and pHelper Onedayafterplatingthecells,co-transfectwithapAAVvector(eitherpAAV-GOIvectorfromVI-2,pAAV-CRE,orpAAV-LacZ),pRCvector,andpHelpervector.

Fortransfection,theCalPhosMammalianTransfectionKit(Cat.#631312)orXfectTransfectionReagent(Cat.#631317)arerecommended;protocolexamplesforeachkitareprovidedbelow.

a. CalPhosMammalianTransfectionKit(Cat.#631312)ThefollowingprotocolismodifiedfromtheprotocolrecommendedforCalPhosMammalianTransfectionKit.FollowtheprotocolprovidedbelowtoobtainahightiterofAAVsolution.

1. Bring2XHEPES-BufferedSalinetoroomtemperature.2. Dilute2McalciumsolutionwithsterileH2O(includedinthekit)toobtaina333mMcalciumsolution(6-folddilution),andbringtoroomtemperature.

3. MixtheplasmidDNAandcalciumsolutionasfollows:

pAAVVector 1μg/μl 6μlpRCVector 1μg/μl 6μlpHelperVector 1μg/μl 6μlCalciumSolution 333mM 1,000μlTotal 1,018μl

4. Addanequalvolumeof2XHEPES-BufferedSalineatroomtemperature.Closethelidofthetubeandvigorouslyshake15timestomix.

5. Allowtostandfor3min.Note: Adheretoastrict3-minincubationtime,thenproceedquicklyto

thenextstep.Withlongerincubation,largecalciumphosphate-DNAcomplexeswillformandtransfectionefficiencywilldecrease.

6. AddthemixturedropwisetotheculturedAAVpro293Tcells(fromStepVI-3)andculturethecellsfurther.Note: WiththeCalPhosMammalianTransfectionKit,itispossibletocheck

forcalciumphosphatecomplexesusingamicroscope.

b. XfectTransfectionReagent(Cat.#631317)1. VortextheXfectPolymer.2. MixtheXfectReactionBufferandtheplasmidDNA,andvortexvigorouslyfor5sec.

pAAVVector 1μg/μl 13μlpRCVector 1μg/μl 13μlpHelperVector 1μg/μl 13μlXfectReactionBuffer 561μlTotal 600μl

3. Add11.7μlofXfectPolymertotheplasmidmixture,andvortexvigorouslyfor10sec.

4. Allowtostandfor10minatroomtemperature.5. Centrifugethesolutionbriefly.AddthesolutiondropwisetotheculturedAAVpro293Tcells(StepVI-3)andculturethecellsfurther.

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VI-5. Change Culture MediumAtleast6hoursaftertransfection(upto25hours),completelyreplacetheculturemediumwithfreshDMEMcontaining2%FBS.

VI-6. Collection of AAV Particle-Producing Cells (2 - 3 Days after Transfection)1. Add1/80volumeof0.5MEDTA(pH8.0)toaculturemediumcontainingAAV-producingcellsandmixwell.Allowtostandatroomtemperaturefor10min.

2. Collectthedetachedcellsinasterile15-mlcentrifugetube.3. Centrifugeat1,750gat4℃for10min.Completelyremovethesupernatantandcollectthecellpellet.Note: Confirmthatthesupernatanthasbeencompletelyremovedbefore

proceeding;viralparticleisolationmaybeaffectedbyresidualsupernatant.

VI-7. Isolation of AAV Particles from AAV-Producing CellsTheuseoftheAAVExtractionSolutionincludedinthekitisstronglyrecommended.ThismethodyieldsAAVparticleswithhigherpurityandtiterthanstandardfreeze-and-thaworsonicationmethods(VIII.ReferenceData).

1. Loosenthecellpellet(fromstepVI-6)bytappingorvortexingthetube.Note: Ifthecellpellethasnotbeenloosenedsufficiently,theefficiencyofextrac-

tionmaydecrease.Confirmthattherearenoclumpsofcellsbeforepro-ceeding.

2. Add0.5mlofAAVExtractionSolutionA.3. Suspendthecellpelletbyvortexingfor15sec.4. Allowtostandatroomtemperaturefor5min.Vortexfor15secagain.5. Centrifugeat2,000-14,000gat4℃for10mintoremovecelldebris.

Note: IfthetiteroftherecoveredAAVvectorislow,theefficiencymaybeincreasedbyrepeatingsteps3-5.

6. Collectthesupernatantinanewsterilecentrifugetubeandadd50μlofAAVExtractionSolutionBandmixbypipettingtoprepareAAVsolution.Note 1: TheAAVsolutioncanbestoredat-80℃.Thawquicklyina37℃water

bathbeforeuse.Note 2: ThesupernatantmaychangetoapinkcolorafterAAVExtractionSolution

Bisadded.

VII. Measurement of Virus TiterVirustitercanbemeasuredbyreal-timePCR(vectorgenomeassay)orbyinfectionassay(biologicaltitermeasurement).Real-timePCRanalysisofvectorgenomesprovidesrapidquantification,whereasdeterminingtiterbyinfectionintocellsisgenerallymoreaccuratetodetermineinfectiousvirustiter.ThereareothertitrationmethodsforAAVvectorsthatinvolveassayofviralcapsidproteins,butthesemethodsmaydetectnonfunctional(empty)particles.

Vector Genome AssayTheAAVproTitrationKit(forRealTimePCR)Ver.2(Cat.#6233)canbeusedtomeasurevirustiterbyreal-timePCRanalysisusingtheviralITRdomainasatarget.

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Biological Titer MeasurementThetiterisdeterminedbymeasuringtheexpressionofthegeneofinterest.TheprotocolbelowisatitrationmethodusingaAAV2vectorexpressingthefluores-centproteinZsGreen(pAAV-ZsGreen1Vector(Cat.#6231)).1. Preparetargetcellsatadensityof2-4x104cells/mlinDMEMwith10%FBS.2. Inoculateseveralwellsofa24-wellplatewith0.5mlofthecellsuspensionandcultureovernight.

3. PrepareserialdilutionsofthepreparedAAV2particlesolutionusingDMEMwith10%FBSandtheninfectthecellwiththedilutedvirussolution.Thedilutionratiodependsonthevirustiter,butserialdilutionsinthe1,000-100,000-foldrangearerecommended.

4. Threedaysafterinfection,detachthecellsusingTrypsin/EDTA,andanalyzeZsGreenexpressionbyflowcytometry.

VIII. Reference Data VIII-1. Increase in AAV2 Titer by the pRC2-mi342 VectorThepRC2-mi342vectorinthekit(Cat.#6230,6652,6655)canbeusedtoproducehightiterrecombinantAAV2particles.

[Methods]VirusproducingCells:HEK293Transfection:CalciumphosphatemethodPlasmids:

•pAAV-CMV-AcGFP1Vector•pRC2-mi342VectororpRC2Vector*•pHelperVector

Culture:T25Flask*pRC2Vector: Vectorlackingthehsa-miR-342expressioncassette

AAV2particleswereextractedandthetiterwasevaluatedbyreal-timePCR.

Figure8.EffectofmiRNA-342onAAV2production.

pRC2 pRC2-mi342

Totalvectorgenomes(vg)

4.5x1010

05.0x1091.0x10101.5x10102.0x10102.5x10103.0x10103.5x10104.0x1010

3.86x1010

1.89x1010

[Results]ThepRC2-mi342vectorresultedinatwo-foldincreaseintiter(vectorgenomes)ascomparedtopRC2.

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VIII-2. Efficiency of AAV Particle Extraction Using AAV Extraction SolutionTheAAVExtractionSolutionsAandBinthissystemcanbeusedtoeasilyandefficientlyextractAAVparticlesfromAAV-producingcells.

A. Comparison with the Freeze-Thaw Method; Virus YieldHEK293cellsweretransfectedwiththepAAV-ZsGreen1vector(Cat.#6231)andotherplasmidsforAAV2.AAV2particlesexpressingZsGreen1wereextractedfromthecellsusingeitherAAVproExtractionSolutionorthefreeze-and-thawmethod.Thetiteroftheviralextractwasdeterminedusingthevectorgenomeassay(Figure9A)andbiologicaltitermeasurementwithHT1080cells(Figure9B).TheinfectiousAAV2viruscanbeobtainedeasilyandefficientlyusingAAVproExtractionSolution.

Genomictiter(vg/ml)

8.0 × 1010

0

1.0 × 1010

2.0 × 1010

4.0 × 1010

5.0 × 1010

6.0 × 1010

7.0 × 1010

9.0 × 1010

3.0 × 1010

Figure9A. AAV2extractionefficiencyusingAAVproExtractionSolution(vectorgenomeassay).(vg:vectorgenome)

ZsGreen1(%)

Figure9B. AAV2extractionefficiencyusingAAVproExtractionSolution(biologicaltiter).

AES: AAVproExtractionSolutionF/T: Freeze-thawmethod

AES F/T0

5

10

15

20

25

30

35

AESF/T

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B. Comparison with Freeze-Thaw Method; PurityAAV2particleswereobtainedfromHEK293producercellsusingAAVproExtractionSolutionorthefreeze-thawmethod.TheamountofviralgenomicDNAineachAAV2extractwasquantifiedbyreal-timePCR.Then,theequivalentof1x109vgofeachAAV2extractwasanalyzedbySDS-PAGEtoevaluatetheamountofproteinimpurity(Figure10).Inaddition,residualcellulardsDNAcontentineachAAV2extractwasassayedusingtheintercalationmethod(Figure11).TheresultsindicatethattheuseoftheAAVproExtractionSolutionclearlyreducedtheamountofproteinimpuritiesanddsDNAincomparisonwiththefreeze-thawmethod.

AES:AAVproExtractionSolutionF/T:Freeze-and-ThawMethod(1x109vg/lane)

Figure10. SDS-PAGEofAAV2extract.ThepurityofAAV2preparedusingtheAAVproExtractionSolution(AES)wasfargreaterthanthepurityofAAV2preparedusingthefreeze-thawmethod(F/T).

Figure11. dsDNAinAAV2extracts.ResidualdsDNAwaslesswhenAAV2waspreparedusingtheAAVproExtractionSolution(AES)ascomparedtothefreeze-thawmethod(F/T).

μg/1x1012 vg

Freeze-ThawMethod

AAVproExtractionSolution

1009080706050403020100

92.21

7.77

F/TAESAAV2

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VIII-3. Infection with AAV2-CRE Particles AAV2-CREviralparticleswerepreparedusingtheAAVproHelperFreeSystem(AAV2-CRERecombinase)(Cat.#6652)andpurifiedusingtheAAVproPurificationKit(AAV2)(Cat.#6232).ParticleswereusedtoinfectHEK293cellsthatareengineeredtofluoresce(ZsGreen1)whenrecombinationoccurswithCrerecombinase.TheproportionoffluorescentcellscorrelatedpositivelywiththeamountofAAV2-Creparticlesusedforinfection.

Figure12.FACSanalysisofHEK293cellsinfectedwithAAV2-CREparticles.

Uninfected(0.07%)

100vg/cell(30.81%)

12,500vg/cell(92.05%)

100

80

60

40

20

0101 102 103 104 105

ZsGreen1

%ofMax

VIII-4. Infection with AAV2-LacZ ParticlesAAV2-LacZviralparticleswerepreparedusingtheAAVproHelperFreeSystem(AAV2-LacZ)(Cat.#6655)andpurifiedusingtheAAVproPurificationKit(AAV2)(Cat.#6232).ParticleswereusedtoinfectHT1080cells.StainingwasperformedusingtheBeta-GalactosidaseStainingKit(Cat.#631780).

Figure13.X-galstainingofHT1080cellsinfectedwithAAV2-LacZparticles.

AAV2-LacZNC

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IX. References1) Miyake,etal .JNipponMedSch.(2012)79(6):394-402.2) VanVliet,etal. MethodsMolBiol. (2008)437:51-91.3) Wu,etal .MolTher. (2006)14(3):316-27.4) Zincarelli,etal .MolTher. (2008)16(6):1073-80.5) Ellis,etal.VirolJ .(2013)10:74.

X. Related ProductspAAV-ZsGreen1Vector(Cat.#6231)AAVpro®PurificationKit(AllSerotypes)(Cat.#6666)AAVpro®PurificationKit(AAV2)(Cat.#6232)AAVpro®TitrationKit(forRealTimePCR)Ver.2(Cat.#6233)AAVpro®ExtractionSolution(Cat.#6235)AAVpro®PackagingPlasmid(AAV2)(Cat.#6234)CalPhos™MammalianTransfectionKit(Cat.#631312)Xfect™TransfectionReagent(Cat.#631317/631318)AAVpro®293TCellLine(Cat.#632273)Beta-GalactosidaseStainingKit(Cat.#631780)

AAVproisaregisteredtrademarkofTAKARABIOINC.In-FusionisaregisteredtrademarkofTakaraBioUSA,Inc.CalPhosandXfectaretrademarksofTakaraBioUSA,Inc.

NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTAKARABIOINC.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656973orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

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Cat. #6230, 6652, 6655v201611Da